This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Post-transcriptional and post-translational modification of ribosomal RNA and proteins is common in most or-ganisms, but the function of many of these modifications is not known. In bacteria, loss of some modifications leads to increased or decreased resistance to antibiotic drugs. We investigate the significance of these modi-fications for ribosome structure and function. This project is carried out in collaboration with Albert Dahlberg at Brown University, Venki Ramakrishnan at the MRC, UK, and Frank Murphy at the APS. We have now produced crystals for 30S ribosomal subunits from T. thermophilus with the gene for the rRNA methyltrans-ferase KsgA deleted. The KsgA dimethyltransferase modifies residues A1518 and A1519 of the 16S rRNA. This modification appears to be conserved in all organisms and confers sensitivity to the antibiotic kasu-gamycin. Structural information for the unmodified 30S subunits will lead to a better understanding of the function of these modifications. In a second approach, we are studying the impact of mutations on ribosome structure. We have produced 30S subunit crystals for two mutant forms: the streptomycin-dependent mutant G524U and the reverting double mutant G524U / A10G. We plan to determine structures of the mutant 30S ribosomal subunits in the apo-form and in complex with streptomycin to understand the rearrangements that lead to the altered binding of the antibiotic in the mutant forms.